Detailed Protocol to Perform Direct PCR Using Filamentous Fungal Biomass—Tips and Considerations

The precise and rapid detection of fungi is important in various fields, including clinics, industry, and agriculture. While sequencing universal DNA barcodes remains the standard method for species identification and phylogenetic analysis, a significant bottleneck has been the labor-intensive and time-consuming sample preparation for genomic DNA extraction. To address this, we developed a direct PCR method that bypasses the DNA extraction steps, facilitating efficient target DNA amplification. Instead of extracting genomic DNA from fungal mycelium, our method involves adding a small quantity of mycelium directly to the PCR mixture, followed by a heat shock and vortexing. We found these simple adjustments to be sufficient to lyse many filamentous fungal cells, enabling target DNA amplification. This paper presents a comprehensive protocol for executing direct PCR in filamentous fungi. Beyond species identification, this direct PCR approach holds promise for diverse applications, such as diagnostic PCR for genotype screening without fungal DNA extraction. We anticipate that direct PCR will expedite research on filamentous fungi and diagnosis of fungal diseases. Key features • Eliminates the time-consuming genomic DNA extraction step for PCR, enhancing the speed of molecular identification. • Adds a small quantity of mycelium directly into the PCR mix. • Emphasizes the crucial role of heat shock and vortexing in achieving efficient target DNA amplification. • Accelerates the molecular identification of filamentous fungi and rapid diagnosis of fungal diseases.

The precise and rapid detection of fungi is important in various fields, including clinics, industry, and agriculture.While sequencing universal DNA barcodes remains the standard method for species identification and phylogenetic analysis, a significant bottleneck has been the labor-intensive and time-consuming sample preparation for genomic DNA extraction.To address this, we developed a direct PCR method that bypasses the DNA extraction steps, facilitating efficient target DNA amplification.Instead of extracting genomic DNA from fungal mycelium, our method involves adding a small quantity of mycelium directly to the PCR mixture, followed by a heat shock and vortexing.We found these simple adjustments to be sufficient to lyse many filamentous fungal cells, enabling target DNA amplification.This paper presents a comprehensive protocol for executing direct PCR in filamentous fungi.Beyond species identification, this direct PCR approach holds promise for diverse applications, such as diagnostic PCR for genotype screening without fungal DNA extraction.We anticipate that direct PCR will expedite research on filamentous fungi and diagnosis of fungal diseases.

Background
Polymerase chain reaction (PCR) is a powerful tool widely used to amplify specific target regions of DNA.Since its invention in the mid-1980s [1,2], PCR has become a fundamental technique employed in various fields.A notable application of PCR is in the diagnosis of pathogenic diseases.It allows for the amplification of universal DNA barcodes, facilitating the identification of causative pathogens [3,4].Given that multiple copies of DNA are generated from the DNA template, the preparation of this template is essential for performing PCR.For filamentous fungi, the steps for DNA extraction and purification have been prerequisites for PCR amplification.Most fungi possess a rigid cell wall, predominantly constituted of polysaccharides like chitin and glu cans [5,6].This results in inefficient fungal DNA extraction.The procedure for extracting DNA from fungal sources requires laborious and time-consuming steps involving the grinding of freeze-dried mycelia [7].Additionally, the fungal DNA extraction process involves multiple stages using toxic chemicals like phenol and chloroform.A technique known as colony PCR involves directly adding cells into the PCR mixture, and it has found widespread application in bacterial and yeast research [8].In the case of filamentous fungi, previous studies have suggested various PCR methods to minimize fungal DNA extraction steps.The methods proposed by Alshahni et al. and Walch et al. eliminated the need for fungal genomic DNA extraction [9,10].However, these methods incorporated additional steps, such as using lysis buffer or bovine serum albumin (BSA), to lyse fungal cell walls.Despite these advancements, the adoption of these methods for filamentous fungi has been limited due to their reduced PCR efficiency and the need for a cell lysis process.Here, we introduce a direct PCR procedure tailored for filamentous fungi.This approach eliminates the need for the fungal DNA extraction process and the use of additional reagents such as BSA or proteinase.Our method involves adding a small amount of mycelium directly into the PCR mixture, followed by heat shock and vortexing.These heat shock and vortexing steps are key in breaking the fungal cell walls and membrane, releasing the genomic DNA for PCR amplification.This method will expedite the molecular identification of filamentous fungi and facilitate rapid diagnosis of fungal diseases.

Laboratory supplies
Published: Nov 05, 2023 3. Gently scratch the fungal culture using a pipette tip to collect a small amount of mycelium (Figure 1).

D. Purification and sequencing
1. To purify the DNA samples, use MEGAquick-spin TM Plus Total Fragment DNA Purification kit and follow the protocol provided with the kit.2. Transfer 50 μL of your PCR product into a 1.5 mL microcentrifuge tube and add 250 μL of agarose gel lysis buffer (included in the DNA purification kit).Mix well by vortexing.3. Insert a spin column in a collection tube.4. Transfer the sample mixture to the spin column and centrifuge at 11,000× g for 30 s. 5. Discard the flowthrough.6. Add 750 μL of the ethanol-added wash buffer solution (included in the kit) to the spin column and then centrifuge at 11,000× g for 30 s. 7. Discard the flowthrough.8. Centrifuge again at 13,500× g for 3 min to dry the column matrix.9. Place the spin column in a new 1.5 mL microcentrifuge tube.10.Add 20-40 μL of elution buffer (included in the kit) to the membrane center of the spin column and wait for 3 min.11.Centrifuge at 13,500× g for 1 min to elute the DNA.12. Measure DNA concentration using a NanoDrop 2000 and submit the samples to a sequencing service center.

Data analysis
DNA sequences were used to conduct a BLAST search against the NCBI GenBank database for species identification.
This protocol or parts of it has been used and validated in the following research article: • Jeon et al. (2023) [4].Application of direct PCR for phylogenetic analysis of Fusarium fujikuroi species complex isolated from rice seeds.Frontiers in Plant Science (Figures 1, 2, and 3).F. graminearum strain Z-3639 was subjected to amplification of the TEF-1α region using a direct PCR approach.The different quantities of mycelium affected PCR efficiency as follows: PC: positive control (using extracted genomic DNA), NC: negative control (no template control), S: small amount of mycelium (as shown in Figure 1), M: medium amount (twice that of S), and L: large amount (twice that of M).

General notes and troubleshooting
2. While direct PCR can be performed using various PCR enzymes, we strongly recommend using the Taq PCR Premix.This premix contains all necessary components for the reaction (including polymerase, dNTP, reaction buffer, and loading dye), except for the template DNA and primers.Our observations show that Taq PCR Premix offers a notably higher success rate compared to other PCR enzymes.Furthermore, the presence of a loading dye in the Taq PCR Premix improves visualization of the added mycelium, allowing for easier verification of mycelium quantity.3.In the previous study [4], direct PCR amplification of the ITS region was tested on samples from major fungal lineages including Ascomycota, Basidiomycota, and Mucoromycota.Our method displayed a remarkable PCR success rate of 92% (34 out of 37 fungal species).Nonetheless, the ITS region of three species-Mucor mucedo, Aspergillus niger, and Aspergillus brasiliensis-remained unamplified by direct PCR.Notably, these fungi exhibited highly hydrophobic mycelium and spores, which likely inhibited cell lysis within the PCR mixture.4. In addition to the ITS, the TEF-1α region and other genotype markers were successfully amplified using the direct PCR method to identify strains within the Fusarium fujikuroi species complex. 5.If the PCR efficiency is low, consider increasing the duration of vortexing after the heat shock step.6.It is advisable to include a reaction containing isolated genomic DNA as a positive control in each experiment.
Also, always have a no-template control.This approach can provide valuable insights when troubleshooting is necessary.

Figure 1 .
Figure 1.Mycelium collection for direct PCR.Aerial hyphae of Fusarium graminearum were collected using a pipette and 10 μL pipette tips.The distal part of the pipette tip is magnified for clarity.Scale bar = 500 μm.

7 Published: Nov 05, 2023 1 .
Cite as: Jeon, H. et al. (2023).Detailed Protocol to Perform Direct PCR Using Filamentous Fungal Biomass-Tips and Considerations.Bio-protocol 13(21): e4889.DOI: 10.21769/BioProtoc.4889.The amount of mycelium added can have a significant effect on the PCR outcome.Always ensure that only asmall quantity of mycelium is added to the PCR mixture.Introducing too much mycelium may lead to reaction blockages and potential failure (Figure2).

Figure 2 .
Figure2.Influence of mycelium quantity on direct PCR efficiency.F. graminearum strain Z-3639 was subjected to amplification of the TEF-1α region using a direct PCR approach.The different quantities of mycelium affected PCR efficiency as follows: PC: positive control (using extracted genomic DNA), NC: negative control (no template control), S: small amount of mycelium (as shown in Figure1), M: medium amount (twice that of S), and L: large amount (twice that of M).